Method for determining the allelic state of the 5&#39;-end of the $g(a)s1- casein gene

ABSTRACT

The invention refers to a genetic marker at the 5′-flanking region of the alpha-S1 casein gene (CSN1S1) and the casein gene complex as well as a method to classify cattle, independent of age and lactation, through determination of the allelic state within this area as well as the application of this method to select organisms with a preferred allele, for instance in the marker-supported selection.

The invention refers to a genetic marker at the 5′-flanking region ofthe αS1 casein gene (CSN1S1) and the casein gene complex as well as amethod to classify cattle, independent of age and lactation, throughdetermination of the allelic state within this area as well as theapplication of this method to select organisms with a preferred allele,for instance in the marker-supported selection.

TECHNICAL STATE OF THE ART

The hereditary potential of breed animals (regarding the milk proteincontent and other characteristics relevant to the breeding) is estimatedat present through estimating the breeding value based on test matingsand performance records of the descendants. The disadvantage of thisconventional procedure is obvious. For cattle, it takes approx. 3 yearsfrom the first insemination by a test bull until the first daughtersbegin lactating, thus approx. 4 years until the registration of acomplete lactation of the daughter. Only thereafter can the breed valuebe estimated. Until then, from both maintaining the bulls until thefirst estimated breeding values are available and the test mating, costsarise, which are substantial during this long period and due to thetotal amount of animals. This applies analogically to the registrationof the own contribution and the determination of a breed value of cows.

Therefore, for some years, with the help of the progress in the genomeanalysis, international efforts have been undertaken to develop geneticmarkers and direct gene tests to identify performance parametersrelevant to breeding. Hereby, genome-wide analysis of markers have madepossible, with the help of the linkage analysis, the approximation tothe chromosome range, in which the gene locations are situated, whichare relevant to the determination of performance, the so-called QTLregions (QTL=quantitative trait loci). Such QTL studies and theresulting tests are described among others in the WO 20000 36143 and theWO 2001 57250 A2/A3. Further details of the QTL analysis of farm animalsas well as a procedure based on QTL studies to also isolate causalcandidate genes, are described in DE 100 17 675 A1. The disclosure ofthe invention is included therein.

Regarding cattle and other species bred for milk production, the crucialcriteria are the milk quantity, the protein content and fat. For thesecriteria, different QTL were identified, among other locations on thechromosome BTA 6. The potential QTL regions for protein contents areindicated relatively uniformly from different working groups within thearea around or between the micro satellite markers BM143 and TGLA37 andthus approximately 20-30 centimorgans (cM) away from the casein locus(Spelman et al. 1996, Genetics 144, 1799-1808; Georges et al. 1995,Genetics 139, 907-920; Boldly et al. 1996, J Anim Breed Genet 133,355-362; Zhang et al. 1998, Genetics 149, 1959-1973). According toNadesalingam et al. (2001, Mammalian Genome 12, 27-31) the casein genesare, however, as well excluded as candidates for the observed QTLeffects due to their position (40 cM away from the QTL).

Since the mid 80's, genetically conditioned milk protein varieties ofcattle have been analysed with regard to an influence on milk yield andquality characteristics. This was realized partly by registering (in themilk) the phenotipically distinguishable protein variants (Ng-Kwai-Hanget al., 1984, J Dairy Sci 67, 835-840 and Ng-Kwai-Hang et al., 1986, JDairy Sci 69, 22-26,), later by means of molecular genetic processes,which provided evidence of the genetic mutations upon which the proteinvariants are based (Sabour et al., 1996, J Dairy Sci 79, 1050-1056).Studies up to now partly reveal conflicting results of the examinedvariants which can not always be confirmed for different breeds andregional origins (summarised by Prinzenberg, 1998, ISBN 3-922306-68-3,chapter 2.4, p. 14-21). The majority of these studies are concentratedon variants of the β-lactoglobulin, and the β- and κ-casein, since inthe αs1 casein, the frequency of only two protein variants is worthmentioning and in particular, in the already strongly selected milkbreeds like Holstein Friesian/German Holstein, the protein variant αs1casein B can be almost exclusively found (Ng-Kwai-Hang et al., 1990, JDairy Sci 73, 3414-3420; Erhardt et al., 1993, J Animal Breed Genet 36,145-152; Lien et al., 1999, Animal Genetics 30, 85-91). In a more recentstudy of dairy cattle with different proportions of Holstein blood(Freyer et al., 1999, J Animal Breed Genet 116, 87-97), αs1 casein wasalso not used in the linkage analysis due to the lack of variability.

Various tests are described concerning the molecular geneticdifferentiation of the αs1 casein variants B and C (David & Deutch 1992,Animal Genetics 23, 425-429; Schlee & Rottmann 1992, J Anim Breed Genet109, 316-319). Individual gene test procedures for the rare alleles A, Dand F also exist, (Prinzenberg 1998, ISBN 3-922306-68-3; chapter 4.1, p.61-71), as well as for the proof of a quantitative variant of the αs1casein G (Mariani et al 1995, L'industria del Latte 31, 3-13). By meansof sequencing around 1,000 base pairs (bp) from the 5′-region of the αs1casein gene from various cattle breeds, Schild & Geldermann (1996)showed 17 variable positions in the 5′-flanking region of the CSN1S1gene, of which 5 have been detected due to different recognitionsequences for the restriction endonucleases with Polymerase ChainReaction-Restriction Fragment Length Polymorphism (PCR-RFLP). Accordingto Ehrmann et al. (1997, J Animal Breed Genet 114, 121-132), the5′-flanking variants are each linked with certain protein alleles, insuch a manner that the existence of given protein variants implies theexistence of certain variants in the 5′-flanking region. Koczan et al.(1993, Animal Genetics 24, 74) also described a gene test todiscriminate the αs1 casein B against C in German American Holstein,Black and White, and Jersey cows which is based on a fragment from the5′-flanking region of the αs1 casein. For the last test mentioned, thestrict linkage with the protein mutations αs1 casein B and C has howeverin the meantime been refuted and therewith, the validity for thefollowing breeds: Aberdeen Angus, Anatolian black, Angeln, AsturianValley, Ayrshire, British Frisian, Casta Navarra, Charolais, Chianina,Fighting Bull, Hereford, Jersey, Maremmana, Pezzata Rossa, Piedmontese,Scottish Highland, Turkish Grey Steppe (Jann et al., 2001; Arch. Tierz,Dummerstorf 45, 13-21).

The casein genes are mapped as a closely linked gene locus in cattle andsheep at chromosome 6, in humans in chromosome 4, and in mice atchromosome 5. The linking of casein genes has also been proven for otheranimal species (rabbit, pig, goat). Due to this close linkage, thepresent designation of the site of the αs1 casein gene in the geneticmap of cattle is linked to the site of the κ-casein gene. In theup-to-date gene map for cattle, the physical position BTA6q31-33, andthe genetic position 82.6 cM (MARC97) and 103.0 cM (IBRP97) resp., arestated for both genes. For this reason, the recombination rate betweenthe αs1 casein and κ-casein gene is assumed to be zero.

The utilization of a lactalbumin sequence for the selection of breedinganimals is revealed in EP0555435. Likewise, for the bovine κ-casein,there exist numerous gene tests (Denicourt et al., 1990, Animal Genetics21, 215-216; Medrano & Aguilar-Cordova, 1990, Biotechnology 8, 144-145;Pinder et al., 1991, Animal Genetics 22, 11-20; Schlee & Rottmann, 1992,J Animal Breed. Genet. 109, 153-155; Zadworny & Kuhnlein, 1990, Theor.Appl. Genet. 80, 631-634), since influences on the processingcharacteristics and cheese producing ability of the milk are attributedto this protein (see Lodes et al., 1996, Milchwissenschaft 51, 368-373and 543-548).

Due to the mammary gland-specific expression, the promoters of bovinemilk protein genes and also the αs1 casein gene are utilized in thecreation of transgenic animals and for expression in cell cultures. DE38 54 555 T2, the content of which is referred to here, describes theutilization of the αs1 casein promoter and signal peptide for theproduction of recombinant proteins in the milk of mammals. Rudolf alsogives an overview of the use of transgenic animals for the production ofrecombinant proteins and the promoters used for this purpose. (1999,Trends in Biotechnology (TIBTECH) 17, 367-374).

Winter et al. (2002, PNAS 99, 9300-9305) describe a direct gene test fora gene from the fatty acid metabolism (DGAT1) and attribute an effect onthe milk fat content to this gene.

The disadvantage of all procedures which phenotypically differentiate(namely within milk samples) the genetic variants, based on a milksample, lies in the fact that lactating cows can be studied exclusively.Thus, there exists the need to be able to examine the animal independentof lactation. Furthermore, the proven polymorphisms in the region of themilk protein gene to be coded do not represent a reliable marker formilk performance traits according to the current state of the art. Theexisting QTL analyses point to a QTL 1 which lies outside the milkprotein genes. For αs1 casein, there is currently no marker availablewith sufficient variability, causing the fact that this gene's effectson lactating performance and content characteristics can almost not bestudied. All available test procedures depend on the molecular geneticdifferentiation of the also phenotypically available variation.

The micro satellite markers, which was ascertained through QTL analyses,are only suited for conditional use in the marker protected selection,because the respective marker-QTL-linkage must first be explained. Ineach case, with these microsatellite markers it is a matter of indirecttests which, depending on the closeness of the linkage to the causalgene location, have less reliable results. The disadvantage of theprocedures in EP 0555435 lies in the fact that α-lactalbumin only makesup a small portion (ca. 2-5%) of the entire milk protein. At approx.80%, the caseins (αs1-, αs2-, β- and κ-casein) form the largest portionof the total protein. Thus, by applying these selection markers, onlyminor breeding progress is to be expected.

The gene test for DGAT1 from Winter et al. has the disadvantage that,from the perspective of the breeder and milk producer, the milk fatcontent is not of primary interest, but rather takes second place behindthe protein content.

The disadvantage of the procedures in DE 38 54 555 T2 lies in the factthat the utilized portion of the αs1 casein promoter is not more closelycharacterized by means of a nucleotide sequence. A 9 kb fragment isutilized containing exons I and II, terminated with recognition sitesfor KpnI and BamHI. No consideration of the exact base effect orpossible variations takes place which can influence the effectiveness ofthe expression with this segment of the promoter.

Currently, there are no reliable markers for milk protein content and nodirect genetic test for a functional gene segment in order to test ananimal's genetic potential, independent of age and lactation.

PROBLEMS OF THE INVENTION

Hence, it is the problem of the invention to make available a geneticmarker and a procedure for the classification of milk production traitsin order to examine an animal's milk production traits by means of theirgenetic material, independently of age and lactation.

This problem is solved by making available, within the region of the αs1casein gene, a marker which remains polymorphic and genetic also withinselected milk breeds, and through a procedure which enables theclassification of the animals independently of age and lactation, thegenetic mapping of the αs1 casein gene, the examination of effects whichare either closely linked with this gene location or thereby directlycaused, as well as a breeding utilization.

Based on the invention, the procedure refers to a genetic test for afunctional gene segment, the reliability of the results is greater thanwith linkage markers and the test result is available within a few daysto a few hours, whereby the substantial costs of test mating can bereduced. Thus, the procedure based on the invention eliminates thedescribed disadvantages in the technical state of the art.

With the marker based on the invention the selection of especiallyadvantageous promoters for the production of expression vectors andtransgenic animals is possible.

In an especially advantageous practical embodiment, the inventionconsists of a test kit, which contains the oligonucleotides for theenrichment of a segment of the marker sequence of the αs1 casein gene,preferably the primer 1 CSN1S1pro1f (5′ GAA TGA ATG AAC TAG TTA CC 3′),primer 2 CSN1S1pro1r (5′ GAA GAA GCA GCA AGC TGG 3′) and primer 3CSN1S1pro2r (5′ CCT TGA AAT ATT CTA CCA G 3′) as well as referenceprobes for one or more sequences of the marker sequence of the αs1casein gene and alleles thereof.

The following figures are enclosed with the description:

FIG. 1 DNA-sequence from the 5′-flanking region of the αs1 casein gene,in the following designated as marker sequence

FIG. 2 Alignment of the nucleic acid sequences of the allelic state ofthe αs1 casein gene allele 1, allele 2, allele 3, allele 4 (differencesin potential transcription factor-interfaces are highlighted)

FIG. 3 Schematic representation of the migration pattern of the alleles1 to 4 of the marker CSN1S1 in the analysis SSCP.

FIG. 4 Result of the variance analysis

It was surprisingly discovered that the examined sequence segment, whichis flanked by the oligonucleotides CSN1S1pro1f and CSN1S1pro1r orCSN1S1pro2r (grey box in FIG. 1) within the breed German Holstein,contains four alleles which were detectable through a single-strandconformation polymorphism analysis and thus is sufficiently polymorphicin order to realize a genetic mapping and analysis concerning theeffects of the alleles on the milk performance parameters.

This concerns a fragment of 1061 bp within the 5′-flanking region andthe exon 1 (refer to FIG. 1), in particular the fragment of 654 bp whichis flanked by the two oligonucleotides CSn1S1pro1f and CSN1S1pro1r.

The four alleles were cloned and sequenced. The sequence analysis was inaccordance with the sequence published by Koczan et al. (1991, NucleicAcids Research 19, 5591-56596; Genbank Acc. No. X59856) for allele 2,except for the length of poly-T (from position 390 of FIG. 1 onwards).The alleles 1, 3 and 4 differ from this sequence by varioussubstitutions and deletions. The variable positions are highlighted inthe sequence alignment (FIG. 2). In alleles 1 and 4, potentialtranscription factor-binding sites are each affected by mutations. Thus,in allele 1, two potential binding sites (for AP-1 and YY1) cease toexist, whereas in allele 4, a new potential ABF1-binding site emerges.

The polymorphism found is therefore located in a supposedly functionalgene region and thus, is a suitable marker for milk production traits,in particular for the protein content.

Based on the current invention, the sequence fragment is flanked by thefollowing oligonucleotide sequence, which is utilized as a primer foramplification by means of PCR, whereby the combinations Primer 1 withPrimer 2, and Primer 1 with Primer 3 are possible: Primer 1: CSN1S1pro1f(5′ GAA TGA ATG AAC TAG TTA CC 3′) Primer 2: CSN1S1pro1r (5′ GAA GAA GCAGCA AGC TGG 3′) Primer 3: CSN1S1pro2r (5′ CCT TGA AAT ATT CTA CCA G 3′)

The primer binding sites are shaded grey in FIG. 1.

A procedure, based on the invention, is made available, which can becarried out directly at the hereditary material of the organism to beexamined. With the help of the marker based on the current invention, agenetic mapping of the αs1 casein gene within the linkage map is madepossible and the determination of the allelic condition in individualorganisms, e.g. cattle, is undertaken, which determines within a fewhours the genetic potential with regard to milk protein content.

The procedure for determining the genetic potential with regard to milkprotein content by determining the allelic condition of the marker basedon the current invention, in detail, consists of:

1. Making available the genetic material of the organism to be examined,from male or female breeding cattle or an embryo thereof.

The organism is, by definition, an animal, particularly a mammal, inparticular a bovine, a sheep or a goat, including embryos of thesespecies.

The organism is also a genetically modified organism (GMO), whichcontains the described sequence fragment of the 5′-flanking region(FIG. 1) and of the αs1 casein gene or parts thereof.

The genetic material is, by definition, genomic DNA or RNA from animals,but also plasmid DNA from bacteria, from artificial chromosomes such asBACs and YACs or constructions created from genetic material of variousorganisms for specific applications, e.g. for the production oftransgenic organisms.

The source material for the extraction of material containing DNA or RNAis namely blood, leukocytes, tissue including biopsy material, milk,sperm, hair, several cells including cell material from embryos, abacteria culture or isolated chromosomes. Furthermore, genetic materialalready amplified beforehand, which contains the marker sequence(FIG. 1) or parts thereof, is again source material.

2. Selective isolation or enrichment of the sequence fragments of FIG.1, or a sequence which contains portions thereof, preferably theillustrated sequence fragment from position 1 to 654 of FIG. 1.

The isolation of genetic material is achieved by standard methods asthey are described in the handbook “Molecular Cloning” (Sambrook,Fritsch, Maniatis, 1989; Cold Spring Harbour Laboratory Press, NewYork), or can be carried out by using commercially obtainable kits (e.g.Nucleospin, Machery Nagel, Düren, Deutschland).

The enrichment is achieved preferably by means of polymerasechain-reaction (PCR, Mullis & Falloona, 1987, Methods in Enzymology 155,335-350), whereby fluorescently marked, radioactively marked, orchemically marked primers can also be utilized. When using RNA asgenetic material, a reverse transcription must be carried out beforehand(Myers & Gelfand 1991, Biochemistry 30, 7661-7666).

The sequence fragment is enhanced preferably by the followingoligonucleotide sequences based on the current invention, which areutilized as a primer for the amplification by means of PCR, whereby thecombinations primer 1 with primer 2, and primer 1 with primer 3 arepossible

The sequence fragment is enhanced preferably with the followingoligonucleotide sequences, based on the current invention, as primer forthe amplification, whereby the combinations Primer 1 with Primer 2 andPrimer 1 with Primer 3 are possible: Primer 1: CSN1S1pro1f (5′ GAA TGAATG AAC TAG TTA CC 3′) Primer 2: CSN1S1pro1r (5′ GAA GAA GCA GCA AGC TGG3′) Primer 3: CSN1S1pro2r (5′ CCT TGA AAT ATT CTA CCA G 3′)

The selection of further primers is explicitly possible, which makespossible the amplification of a partial sequence of the sequencedescribed in FIG. 1, within which variable nucleotide positions arelocated in order to differentiate the alleles 1 to 4.

3. Proof of the allelic state in the isolated or enhanced sequencefragment of FIG. 1, preferably within the partial sequence, which isflanked by CSN1S1pro1f and CSn1S1pro1r.

In order to determine the allelic state, various standard techniques areavailable which are well known by the expert: The sequencing accordingto Sanger et al. 1977, through an illustration of single-strandconformation polymorphisms (SSCP, Orita et al. 1989, Genomics 5,874-879), restriction fragment length polymorphisms (RFLP; Botstein etal. 1980, American Journal of Human Genetics 32, 314-331) and PCR-RFLP(Damiani et al. 1990, Animal Genetics 21, 107-114; Medrano &Aguilar-Cordova 1990, Animal Biotechnology 1,73-77), allele-specific PCR(=ARMS, ASPCR, PASA; Newton et al. 1989, Nucleic Acids Research 17,2503-2516; Sakar et al. 1990, Analytical Biochemistry 186, 64-68; David& Deutch 1992, Animal Genetics 23, 425-429), oligonucleotide-ligationassay (=OLA; Beck et al. 2002, J Clinical Mikrobiol 40, 1413-1419),temperature gradient gel electrophoresis (=TGGE, Tee et al. 1992, AnimalGenetics 23, 431-435) and analogical procedures belonging to thetechnical state of the art.

It is suggested to furnish the primers based on the current inventionwith a marker (fluorescent, radioactive or similar) and to determine theallelic state with a sequencing machine, autoradiography orchemiluminescence. If non-marked primers are utilized, the determinationof the allelic state is carried out by illustrating the fragmentsaccording to gel electrophoresis through coloring of the nucleic acids,e.g. with ethidiumbromid (Sambrook et al., 1989) or through thesilver-coloring procedure (Bassam et al 1991, Analytical Biochemistry196, 80-83).

Furthermore, it is possible to utilize different high throughput methodsfor the mutation screening, including the utilization of oligonucleotidearrays (Dong et al 2001, Genome Research 11, 1418-1424), the TaqManprocedure (Ranade et al 2001, Genome research 11, 1262-1268), thefluorescence polarization method, (Chen et al 1999, Genome Research 9,492-498), mass spectrometric method (MALI-TOF; Sauer et al. 2002,Nucleic Acids Research 30, e22). This enumeration is exemplary and isnot to be understood as limited.

The allelic state is hereby to be understood as the existence of acertain nucleotide sequence within the enriched fragment. FIG. 2exemplifies the nucleotide sequence of four different allelic states ofthe marker based on the current invention (FIG. 2, alleles 1, 2, 3 and4).

In the case of sequencing, a comparison with the correspondingnucleotide sequences 1, 2, 3 and 4 in FIG. 2 must be carried out inorder to establish the analogical correlations between the allele types1 to 4. Based on the indicated nucleotide sequences, it is possible fora person familiar with the state of the art to determine the lengths ofthe fragments through a PCR-RFLP analysis or to conceiveoligonucleotides for the detection through allele-specific PCR. Also,the adjustment of the other aforementioned techniques for a mutationscreening can be done by the expert.

Particularly preferable is the illustration of the allelic states bymeans of single-strand conformation polymorphisms (SSCP), as the allelicstate can be read directly from the fragment pattern. In addition to thedetection of the 4 alleles described here, the procedure also enablesthe recognition of further mutations which are not described here. Forthat reason it is also particularly well suited for the analysis of thehomologous genome region of other animal species than cattle. In orderto reduce the duration of the gel electrophoresis, it is recommended toutilize, instead of the entire sequence, a shorter fragment, e.g. thesequence marked with an arrow in FIG. 1 which is defined by theoligonucleotides based on the current invention.

FIG. 4 shows a schematic illustration of the alleles 1 to 4 of themarker CSN1S1 in the SSCP analysis in a 12% acrylamide/bisacrylamide gel(49:1) with a 1% glycerol additive. The fields 1 to 4 represent the fourdifferent separation patterns of the alleles. The migration direction ofthe molecules in the electrical field from the cathode (−) to the anode(+) is represented by an arrow. The single strands of the alleles show atypical, clearly different separation pattern one from another. Since bymeans of silver coloring both DNA single strands are illustrated, eachallele is characterized by two bands.

4. Selection of organisms which carry the respectively preferableallelic state of the marker based on the current invention. This can bee.g. the allelic state 1 or 4, which differs from allele 2 by the amountof potential binding sites for transcription factors.

Practical Embodiments

1. Procedure to Classify the Milk Production Traits ThroughDetermination of the Allelic State of the Marker Based on the CurrentInvention.

Cattle blood is used as source material. The isolation of the geneticmaterial (genomic DNA) is carried out according to the high-salt methodof Montgomery & Sise (1990, NZ J Agric Res 33, 437-441).

In order to realize the amplification of the marker by means of PCRreaction the oligonucleotic sequences based on the current invention areutilized as primers: Primer 1 CSN1S1pro1f (5′ GAA TGA ATG AAC TAG TTA CC3′) Primer 2 CSN1S1pro1r (5′ GAA GAA GCA GCA AGC TGG 3′)

In 15 μl, the reaction solutions respectively contain 20-100 ng genomicDNS to be tested, 10 pmol of each oligonucleotide CSN1S1pro1f andCSN1S1pro1r, 0.5 U Taq DNA polymerase (Peqlab Biotechnologie, Erlangen),50 μM dNTPs in a standard buffer (10 mM Tris-HCl ph 8.8, 50 mM KCl, 1.5mM MgCl₂). The temperature program (of the thermo cycler, model iCyclerof the company Biorad) is selected as follows: 1 min. −93° C. (1×), (40sec −91° C., 40 sec. 57° C., 40 sec −70° C.) (30×) and 3 min −70° C.(1×). Afterwards, cooling to 4° C. takes place.

Afterwards, to each reaction solution, 25 μl of a formamide denaturationbuffer (95% formamid, 0.025% (w/v) bromphenol blue, 0.025% (w/v)xylencyanol FF, 20 mM EDTA) are added respectively, the mixture isheated for 2 min at 93° C., cooled down in ice water and every 4 μl ofthe mixture are loaded onto a 12% acrylamide/bisacrylamide gel (49:1)with a 1% glycerol additive. The separation is carried out over 20 h at420V and 10° C. in a vertical electrophoresis system, utilizing thesquare strip container Penguin P9DS (OWL Scientific, Woburn, USA) with agel of 0.8 mm thickness and a size of 16×16cm. As running and gelbuffer, 0.5× TBE has been utilized. After completion of theelectrophoresis, the gels have been colored with silver nitrateaccording to the protocol of Bassam et al. (1990, AnalyticalBiochemistry 196, 80-83). The development reaction has been stopped bytransferring the gels in an ice-cold 0.04M EDTA solution.

The migration pattern of alleles 1 to 4 is shown schematically in FIG.3. As by means of silver coloring, both (coding and non-coding DNA line)are colored, two fragments per allele are available respectively.

2. Illustration of the Variability of the Marker CSN1S1 in VariousCattle Breeds

From DNA of 83 cattle of the breeds German American Holstein (6 cattle),German Red cattle (4 cattle), Yellow cattle (7 cattle), German Holstein(18 cattle), Black and White (9 cattle), Jersey (13 cattle), Pinzgauer(20 cattle) and Simbrah (6 cattle) the nucleic acid sequence position 1to 655, as mentioned in FIG. 1, is amplified with the oligonucleotides,based on the current invention, CSN1S1pro1f (5′ GAA TGA ATG AAC TAG TTACC 3′) and CSN1S1pro1r (5′ GAA GAA GCA GCA AGC TGG 3′) by means of PCR.The further procedure takes place as described in example 1.

In the breeds examined, the typical separation patterns, as revealed inFIG. 3, appear.

3. Illustration of the Variability of the Marker CSN1S1 Within theGerman Holstein Breed

From blood samples of 503 cows of the breed German Holstein, DNA isisolated according to the method of Mongomery & Sise (1990, NZ J AgricRes 33, 437-441). The enrichment of the sequence given in FIG. 2 isachieved with the primers based on the current invention, as describedabove. The illustration of the existing variations is achieved by meansof SSCP-Technology. Among the cows examined of this breed, all fouralleles are also detectable. The following allele frequencies weredetermined: Allele 1 - 0,031 Allele 2 - 0,739 Allele 3 - 0,194 Allele4 - 0,036

Thus, allele 2 represents the most frequent allele in the GermanHolstein breed, followed by allele 3 and the two rare alleles 1 and 4.

The genotypes occurred in the frequency 22>23>24>12>33>34. The genotypes11 and 14 as well as the combination of these two rare alleles (genotype14) were not found.

4. Genetic Mapping of the Marker CSN1S1

By means of the procedure, based on the current invention, eighthalf-sib families of the breeds German Holstein (7) and Black and White(1) are genotyped with the marker CSN1S1 and subsequently compared withthe results obtained by Thomsen et al. 2000 (J Anim Breed Genet 117,289-306), who already has genotyped these families for 10 furthermarkers on BTA 6 (microsatellite marker) and has established a linkagemap. The genotyping data for CSN1S1 are integrated into this existingdata record. Mapping utilizing the BUILD function of the program packageCRI-MAP (version 2.4, Green et al. 1990, Documentation of CRI-MAP,Washington School of Medicine, St. Louis, Mo., USA) leads to twopossible locations of the CSN1S1 marker: between the markers IL97 andFBN14 or FBN14 and CSN3. The additionally realized FLIPS analysis leadsto a definitive mapping of CSN1S1 between the markers FBN14 and CSN3.The total length of the linkage map of BTA6, calculated with these 11markers, is 161.1 cM. The position of all markers included in thelinkage map, and the corresponding indications from the existing linkagemaps MARC97 and IBRP97, can be seen in the following table 1. Given arethe markers to establish the linkage map of BTA6, the number ofinformative meioses as well as the positions (cM) on the genetic mapcalculated with CRI-MAP (the map based on the current invention isreferred to as “ADR”) in comparison to the two linkage maps MARC97 andIBRP97. For those markers indicated with “n.a.”, no mapping is availablein the respective linkage maps. TABLE 1 Position (cM) Marker InformativeADR MARC97 IBRP97 ILSTS93 193 0.0 0.0 16.0 ILSTS90 156 28.5 11.8 0.0BM1329 141 56.8 35.5 45.0 URB16 228 57.9 n.a. 40.0 DIK82 356 78.5 n.a.67.0 ILSTS097 78 99.6 67.2 89.0 FBN14 187 104.1 n.a. n.a. CSN1S1 280108.1 (like CSN3) (like CSN3) CSN3 102 113.5 82.6 103.0 BP7 208 123.691.2 n.a. BMC4203 186 161.1 112.9 n.a.5. Variance Analysis for Estimating the Effects on the milk ProductionTraits

By means of the procedure based on the current invention a total amountof 729 bulls from 9 half-sib families of the breeds German Holstein andSimmental were genotyped with the marker CSN1S1. The distribution of thegenotypes within the 9 half-sib families is shown in table 2. TABLE 2CSN1S1 Genotype Family n 12 13 14 22 23 24 33 34 1 19 — — — 9 10 — — — 2108 48 5 5 37 9 4 — — 3 106 4 — 3 40 10 37 — 12 4 27 12 3 2 — 10 — — — 512 — 1 — 5 5 — 1 — 6 27 — — — 9 16 1 1 — 7 55 1 — 1 22 4 23 4 8 56 4 2 —26 17 3 1 3 9 319 10 — — 250 50 9 — total 729 79 11 11 398 131 77 3 19

The breeding values of the bulls are centrally estimated by the UnitedInformation Systems Animal Production (Vereinigte InformationssystemeTierhaltung—VIT) in Verden. A total amount of more than 150,000daughters and their performance data are integrated in the estimation ofthe breeding values. From all bulls, deregressed breeding values,concerning the milk yield, the protein and fat yield, the proteincontent (in %) and the fat content (in %), are utilized in the variancecomponent estimation. The deregression of the breeding values is carriedout as described by Thomsen et al. (2001, J Anim Breed Genet. 118,357-370).

The variance component estimation is carried out using the programpackage SAS. First, as unique fixed effect, the marker CSN1S1 isconsidered in the model, because other influence factors (e.g.operational effects, milking frequency) are already corrected in theframe of the estimation of the breeding value and the deregression(influence of the sires). The analysis reveals significant effects ofthe marker CSN1S1 on all studied traits (deregressed breeding values forprotein percentage (DRG_PP), milk yield (DRG_MY1), fat yield (DRG_FY1),protein yield (DRG_PY1), fat percentage (DRG_FP)). Table 3 shows theeffect of CSN1S1 on deregressed breeding values for milk productiontraits, indicating also the probability of error (p) for the effects onthe individual traits. TABLE 3 Trait Probability of error (p) DRG-PP<0.0001 DRG_MY1 0.0011 DRG_FY1 0.0016 DRG_PY1 0.0056 DRG_FP 0.0052

The highest significance is calculated for the effect on DRG_PP. As theexamined marker CSN1S1 is located directly within the regulatory regionof a milk protein gene, this could be an indication of a direct effect.The marker CSN1S1 fulfils the requirements to a functional candidategene.

The highest breeding value for milk (DRG_MY1) is achieved on average bybulls with the genotype 12, whereas the highest breeding values forprotein percentage (DRG_PP) are found within the group with genotype 24.Table 4 shows a compilation of the least square means (LS_means) for thegroups with the genotypes 12, 22, 23 and 24. The table displays theLS_means as well as standard errors for the deregressed breeding valuesfor milk yield (DRG_MY1) and protein percentage (DRG_PP) in groups withdifferent CSN1S1 genotypes. TABLE 4 CSN1S1 LSMEAN ± se type n DRG_MY1DRG_PP 12 79 198.232 ± 15.700 −0.00022534 ± 0.00006470 22 398 155.341 ±6.995 −0.00037495 ± 0.00002921 23 131 138.806 ± 12.192 −0.00038405 ±0.00005271 24 76 112.364 ± 16.007  0.00008175 ± 0.00006650 Alle 684152.353 −0.000307

In order to obtain a more exact clarification, the variance analysis isrepeated within individual families and groups of families withidentical genotypes. Hereby is revealed, that the effect on the milkyield can not be confirmed in all families. In family 9, in which thesires exclusively passed down the allele 2, the only remaining effect isencountered close to the 5% threshold of significance for DRG_PP(p=0.0610). Furthermore, a comparison of the LS_means for the traitsDRG_MY1, DRG_PP, DRG_FP is carried out for all groups of genotypes andwithin each individual family, and it is proved whether the differenceof the LS-means between the genotypes 12, 23 and 24 and the mostfrequent genotype 22 is significant. The results are graphicallyillustrated in FIG. 5.

1. Genetic marker at the 5′-flanking region of the αS1 casein gene(CSN1S1) characterized by the fact that it contains the nucleotidesequence 1-1061, preferably the nucleotide sequence 1-655 at the5′-flanking region of the αS1 casein gene.
 2. Genetic marker accordingto patent claim 1 characterized by its amplification by means of PCRreaction either through Primer 1 CSN1S1pro1f (5′ GAA TGA ATG AAC TAG TTACC 3′) Primer 2 CSN1S1pro1r (5′ GAA GAA GCA GCA AGC TGG 3′)

or through Primer 1 CSN1S1pro1f (5′ GAA TGA ATG AAC TAG TTA CC 3′)Primer 3 CSN1S1pro2r (5′ CCT TGA AAT ATT CTA CCA G 3′)


3. Genetic marker according to patent claim 1 characterized by itsvariability within milk breeds.
 4. Genetic marker according to patentclaim 1 characterized by its utilization in order to determine theallelic state at the 5′-flanking region of the αS1 casein gene. 5.Procedure to determine the allelic state of the 5′-flanking region ofthe αs1 casein gene, characterized by the following steps: a) provisionof the source material of the organism to be examined b) isolation ofthe genetic material c) targeted isolation or enrichment of the markerfragment at the 5′ region of the αs1 casein gene or of a sequence, whichcontains portions of the marker sequence, preferably the fragment 1 to655 of the marker sequence out of the αs1 casein gene d) Proof of theallelic state in the isolated or enriched sequence fragment of themarker fragment of the αs1 casein gene.
 6. Procedure according to patentclaim 5 characterized by the utilization of source material coming froman animal, particularly a mammal, in particular a bovine, a sheep or agoat, including breed animals and embryos of these species.
 7. Procedureaccording to patent claim 5 characterized by the utilization of blood,leukocytes, tissue including biopsy material, milk, sperm, hair,individual cells including cell material from embryos, a bacteriaculture or isolated chromosomes as source material.
 8. Procedureaccording to patent claim 5 characterized by the utilization of sourcematerial coming from a genetically modified organism (GMO) whichcontains the marker fragment of the αs1 casein gene.
 9. Procedureaccording to patent claim 5 characterized by the utilization of geneticmaterial containing genomic DNA or RNA from animals, plasmid DNA frombacteria, from artificial chromosomes such as BACs and YACs. 10.Procedure according to patent claim 5 characterized by achieving theenrichment of the marker segment of the αs1 casein gene by means ofpolymerase chain-reaction.
 11. Procedure according to patent claim 5characterized by the enrichment of the marker segment of the αs1 caseingene by means of polymerase chain-reaction with the oligonucleotidesPrimer 1 CSN1S1pro1f (5′ GAA TGA ATG AAC TAG TTA CC 3′) Primer 2CSN1S1pro1r (5′ GAA GAA GCA GCA AGC TGG 3′) Primer 3 CSN1S1pro2r (5′ CCTTGA AAT ATT CTA CCA G 3′)

as primers, whereby the following combinations are selected: primer 1with primer 2 and primer 2 with primer
 3. 12. Procedure according topatent claim 5 characterized by the determination the allelic state bymeans of SSCP, RFLP, OLA, TGGE, ASPCR, PCR-ELISA, microarray method orthrough nucleic acid sequencing.
 13. Procedure according to patent claim5 characterized by detection of one or more of the allelic states of themarker sequence of the αs1 casein gene.
 14. Utilization of the procedureaccording to claim 5 in order to examine the animals' milk productiontraits, independently of age and lactation.
 15. Utilization of theprocedure according to claim 5 in order to select organisms which carrya certain allelic state or a certain genotype of the marker sequence ofthe αs1 casein gene or a portion thereof.
 16. Utilization of theprocedure according to claim 5 in breeding programs, particularly for amarker-supported selection.
 17. Utilization of the procedure accordingto claim 5 for the selection of increased milk protein yields. 18.Utilization of a marker according to patent claim 1 for genome analysis,in particular to carry out a genetic mapping and/or a linkage analysis.19. Utilization of a marker according to patent claim 1 to createexpression vectors.
 20. Utilization of a marker according to patentclaim 1 to produce transgenic animals.
 21. Testkit, containingoligonucleotides to enrich a segment of the marker sequence of the as Icasein gene, preferably the primer 1 CSN1S1pro1f (5′ GAA TGA ATG AAC TAGTTA CC 3′), primer 2 CSN1S1pro1r (5′ GAA GAA GCA GCA AGC TGG 3′) andprimer 3 CSN1S1pro2r (5′ CCT TGA AAT ATT CTA CCA G 3′) as well asreference probes for one or various sequences of the marker sequence ofthe αs1 casein gene and the alleles thereof.